High-throughput screening assays are a staple of drug discovery, allowing over 100,000 compounds to be screened per day. Targets of high-throughput screens include G-protein coupled receptors, enzymes, hormones, ion channels, nuclear receptors, and DNA transcription factors. In addition to identifying lead compounds (“hits”), drug development requires high-throughput assays to eliminate false positives, validate the target, prioritize the hits, and elucidate structure-activity relationships. The development of high-throughput assays is also required to keep pace with the rapid growth of genomic and proteomic data.
Enzymes that release AMP as a product play a role in a number of diseases. For example, cAMP phosphodiesterases (PDEs) convert cAMP to AMP and, as a result, regulate signal transduction pathways governing vascular resistance, cardiac output, visceral motility, immune response, inflammation, neuroplasticity, vision, and reproduction. PDE inhibitors prolong or enhance cAMP-mediated signaling pathways and have been used in the treatment of pulmonary arterial hypertension, coronary heart disease, dementia, depression, schizophrenia, and other disorders. Other enzymes that release AMP as a product include the ubiquitin ligase and ubiquitin-like ligase proteins, which catalyze the attachment of ubiquitin and ubiquitin-like proteins (e.g. small ubiquitin-like modifier (SUMO) proteins) to their protein substrates, modifying their function or targeting them for degradation by the proteasome. Like proteasome inhibitors, inhibitors of ubiquitin ligases have potential therapeutic value in treating cancer and other diseases by inducing apoptosis. However, since ubiquitin ligases target specific subsets of proteins for degradation, they are more selective than proteasome inhibitors. Enzymes that remove these protein modifications (e.g. deubiquitinating enzymes) also target selected subsets of proteins, providing additional candidates for therapeutic intervention. Lastly, members of the aminoacyl-tRNA synthetase family release AMP as a product during the aminoacylation of tRNA, a crucial step in protein synthesis. Members of this family are potential targets for the development of novel antibiotics and antifungals. For example, an isoleucyl-tRNA synthetase inhibitor, mupirocin, is used to treat multidrug resistant Staphylococcus aureus, while a leucyl-tRNA synthetase inhibitor, Kerydin™, is an antifungal that recently received FDA approval for the topical treatment of onychomycos of the toenails. Furthermore, the essential role that aminoacyl-tRNA synthetases play in protein synthesis makes the human homologs potential targets for chemotherapy agents.
Currently, assays are available to monitor the production of AMP or pyrophosphate, but they either require quenching a reaction to check the AMP/pyrophosphate production a given point in time, and/or they are limited in that they can only be applied to enzymes that form AMP through hydrolysis of ATP. As a result, they cannot be used to follow the activities of enzymes such as phosphodiesterases and ribonucleases, which do not release pyrophosphate as a product. In addition, since the production of inorganic phosphate is being monitored in such assays, they are incompatible with phosphate buffers.
Further, the rise in drug resistant organisms represents a significant world health threat. In the United States, approximately 1.7 million patients acquired an infection while in the hospital, resulting in nearly 100,000 fatalities. Seventy percent of these infections result from antibiotic-resistant bacteria. In addition to increased mortality rates, the rise in antimicrobial resistant organisms increases treatment time and length of stay for hospital patients, hampers medical advancements—including organ transplants, cancer treatment, and surgery—and increases health care costs. Antibiotic resistance adds ˜$20 billion/year to health care costs and results in 8 million additional days that patients spend in the hospital.
Aminoacyl-tRNA synthetases (aaRSes) are essential enzymes, which catalyze the attachment of amino acids to their cognate tRNAs. Several properties of the aminoacyl-tRNA synthetases make them attractive candidates for antimicrobial drugs, including: (1) conservation of the catalytic mechanism across bacterial species, (2) loss of biological fitness in bacteria that are resistant to aminoacyl-tRNA synthetase inhibitors, (3) differences in the catalytic mechanism of bacterial and eukaryotic aminoacyl-tRNA synthetase homologs, and (4) the existence of x-ray crystal structures for all 20 aminoacyl-tRNA synthetases, providing a structural framework for designing inhibitors and elucidating their mechanism of action. Known aminoacyl-tRNA synthetase inhibitors include natural products, such as mupirocin and furanomycin (isoleucyl-tRNA synthetase), borrelidin (threonyl-tRNA synthetase), granaticin (leucyl-tRNA synthetase), indolmycin (tryptophanyl-tRNA synthetase), ochratoxin A (phenylalanyl-tRNA synthetase), and cispentacin (prolyl-tRNA synthetase). In addition, a number of pharmaceutical companies have shown interest in aminoacyl-tRNA synthetase inhibitors as potential therapeutics. For example, the leucyl-tRNA synthetase inhibitor, Keryidin™ (Anacor™ Pharmaceuticals, Palo Alto, Calif.), recently received FDA approval for treatment of onychomycosis (toenail fungus), while methionyl-tRNA synthetase inhibitors developed by GlaxoSmithKline™ have been found to inhibit Trypanosoma brucei infection in mice. Borrelidin, which initially was found to inhibit bacterial threonyl-tRNA synthetases, is currently being tested as a treatment for malaria, suggesting that inhibitors of bacterial aminoacyl-tRNA synthetases may also be useful in treating protozoan parasites. Similarly, cladosporin, which was originally identified as an antibacterial agent, has been shown to inhibit lysyl-tRNA synthetase from the malaria parasite, Plasmodium falciparum. Both the chemical diversity displayed by known aminoacyl-tRNA synthetase inhibitors, and the diversity of their targets, supports the principle that aminoacyl-tRNA synthetases are rich targets for developing novel antimicrobials.
While assays have been developed for monitoring the activity of aminoacyl-tRNA synthetases, a common limitation is their inability to recycle the tRNA substrate. As tRNA is the limiting substrate in these assays, the ability to regenerate it in situ would both increases the sensitivity of the assays, while decreasing the cost of the current technology.